Methods and Compositions for Increasing Protein Production

ABSTRACT

The disclosure provides methods and materials for increasing the expression of a protein of interest such as an antibody by a cell. ABC50 expression or activity is increased which increases expression of the protein or antibody of interest. The disclosure also provides methods and materials for increasing the sensitivity of a cell to an endoplasmic reticulum stress agent such as Econozole by decreasing the level of ABC50.

RELATED APPLICATIONS

The present application is a continuation of copending U.S. patentapplication Ser. No. 13/318,985, filed Nov. 4, 2011, which is a Nationalstage entry of International Application No. PCT/CA2010/000681, filedMay 5, 2010, which claims priority to U.S. Provisional PatentApplication 61/175,642 filed May 5, 2009, each of these applicationsbeing incorporated herein in their entirety by reference.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“10723-P34775US02_SL.txt” (34,155 bytes), submitted via EFS-WEB andcreated on Feb. 26, 2014, is herein incorporated by reference.

FIELD OF THE DISCLOSURE

The disclosure relates to methods and compositions for proteinproduction and specifically to methods and compositions for increasinghybridoma antibody production.

BACKGROUND OF THE DISCLOSURE

ABC50 (aka ABCF1) is a member of the ATP Binding Cassette (ABC) familyof proteins. ABC50 was first identified as a Tumor Necrosis Factorα-inducible gene in synoviocytes¹, and then re-discovered as a proteinthat purifies with the translation initiation factor elF2².Biochemically, ABC50 stimulates formation of complexes between elF2, GTPand Met-tRNA, implicating it in translation initiation and control.ABC50 is a unique member of the ABC family in that it lackstransmembrane domains. Recently Paytubi et al. showed that theN-terminal region was responsible for elF2 binding³. Binding was foundto be regulated by Casein Kinase 2 phosphorylation in this domain.Overexpression of ABC50 into HEK293 cells was not observed to boostprotein expression³.

Econazole (Ec) is an imidazole antifungal that also induces endoplasmicreticulum (ER) stress by promoting ER Ca²⁺ depletion. Ec's mechanism ofaction involves both Ca²⁺ influx blockade and stimulation of ER Ca²⁺release⁴. The latter effect is mediated by reactive oxygen species (ROS)generation at the mitochondria⁵. Some cancer cells are extraordinarilysensitive to Ec^(6,7).

The market for therapeutic proteins is currently on the order of $60Billion worldwide. The largest component of this market is recombinantmonoclonal antibodies but also includes other protein classes such ascytokines, growth factors such as insulin, coagulation factors, vaccinesubunits and therapeutic enzymes. The diagnostic market is similarlyestimated to be $40 Billion worldwide and a significant fraction of thismarket employs recombinant proteins including monoclonal antibodies.Finally, recombinant proteins for research purposes also represent alarge and growing use for recombinant proteins.

It was recently estimated that about half of the 140 recombinantproteins on the market are produced in mammalian cells⁸. Given therequirement for large amounts of protein, particularly in thetherapeutic setting, there is clearly a need for optimizing natural andrecombinant protein production.

SUMMARY OF THE DISCLOSURE

An aspect of the disclosure includes a method of producing a protein ofinterest in a cell comprising increasing the expression or activity of aABC50 protein or a fragment thereof having eIF2 binding activity; andeffecting the expression of the protein of interest.

In an aspect, the disclosure provides a method of producing aheterologous protein of interest in a cell comprising increasing theexpression or activity of a ABC50 protein or a fragment thereof havingprotein synthesis increasing activity and/or eIF2 binding activity; andeffecting the expression of the protein of interest.

In another aspect, the disclosure provides a method of producing anantibody of interest or fragment thereof in a cell capable of expressingan antibody comprising increasing the expression or activity of an ABC50protein or a fragment thereof having protein synthesis increasingactivity and/or eIF2 binding activity.

Another aspect relates to a method of increasing expression of aheterologous protein of interest in a cell expressing the protein ofinterest, comprising increasing the expression or activity of an ABC50protein or a fragment thereof having protein synthesis increasingactivity and/or eIF2 binding activity.

Yet another aspect relates to a method of increasing expression of anantibody of interest in a cell expressing the antibody of interest,comprising increasing the expression or activity of an ABC50 protein ora fragment thereof having protein synthesis increasing activity and/oreIF2 binding activity.

In an embodiment, the expression or activity of ABC50 protein or afragment thereof is increased by introducing a heterologous ABC50polynucleotide encoding ABC50 protein or a fragment thereof operativelylinked to a promoter.

In another embodiment, the expression or activity of ABC50 protein or afragment thereof is increased by contacting the cell with increasingconcentrations of Econozole (Ec), and detecting increased expression oractivity of ABC50 protein.

In an embodiment, the cell comprises a heterologous polynucleotideencoding the protein of interest operatively linked to a promoter.

In another embodiment, the expression or activity of ABC50 protein or afragment thereof is increased and the expression of the protein ofinterest is effected by introducing a vector comprising a polynucleotideencoding ABC50 protein or a fragment thereof, and a heterologouspolynucleotide of the protein of interest, wherein the polynucleotidesare operatively linked to one or more promoters.

In another embodiment, effecting the expression of the protein ofinterest comprises contacting the cell with an inducer that inducesexpression of the protein of interest or induces expression of ABC50.

In a further embodiment, the ABC50 protein comprises SEQ ID NO: 1, 2 or5; or a protein with at least 90%, 95%, 99% or 99.5% identity with SEQID NO:1, 2 or 5.

In an embodiment, the method results in increased specific cellularexpression and/or production of the protein of interest in comparison toa control cell expressing the protein of interest wherein the controlcell does not have increased expression of an ABC50 protein or afragment thereof having protein synthesis increasing activity and/oreIF2 binding activity.

In another embodiment, the method results in increased specific cellularexpression and/or production of the antibody of interest in comparisonto a control cell expressing the antibody of interest wherein thecontrol cell does not have increased expression of an ABC50 protein or afragment thereof having protein synthesis increasing activity and/oreIF2 binding activity.

In a further embodiment, wherein the increase in expression and/orproduction is about 5% to about 10%, about 11% to about 20%, about 31%to about 40%, about 41% to about 50%, 51% to about 60%, 61% to about70%, 71% to about 80%, about 81% to about 90%, about 91% to about 100%,about 150% to about 199%, about 200% to about 299%, about 300% to about499%, or about 500% to about 1000%.

In an embodiment, the cell is a eukaryotic cell selected from a yeast,plant, worm, insect, avian, fish, reptile and mammalian cell. In anotherembodiment, the mammalian cell is a myeloma cell, a spleen cell, or ahybridoma cell. In yet a further embodiment, the mammalian cell is aleukemia cell, such as HL-60; or a hybridoma cell such as Sp2; or achinese hamster ovary (CHO) cell.

The protein of interest or antibody of interest is, in an embodiment, asecreted protein, an intracellular protein, or a membrane protein.

In another embodiment, the protein of interest is an antibody orantibody fragment or derivative thereof. In an embodiment, the antibodyis monoclonal, polyclonal, mammalian, murine, chimeric, humanized,primatized, primate, or human. In another embodiment, the antibody is afragment or derivative thereof selected from antibody immunoglobulinlight chain, immunoglobulin heavy chain, immunoglobulin light and heavychains, Fab, F(ab′)2, Fc, Fc-Fc fusion proteins, Fv, single chain Fv,single domain Fv, tetravalent single chain Fv, disulfide-linked Fv,domain deleted, minibody, diabody, a fusion protein of one of the abovefragments with another peptide or protein or Fc-peptide fusion.

In an embodiment, the method further comprises isolating the protein ofinterest or the antibody of interest. Where, for example, the protein orantibody of interest is secreted, the method in an embodiment, furthercomprises isolating the secreted protein or secreted antibody ofinterest. Where, for example, the protein or antibody of interest isintracellular, the method further comprises in an embodiment, lysing thecell and isolating the intracellular protein or antibody of interest. Inanother embodiment, where the protein or antibody of interest ismembrane or surface bound, the method in an embodiment, furthercomprises solubilizing the cell membrane and isolating the membraneprotein or surface antibody of interest.

A further aspect provides a process for the production of a protein ofinterest comprising: culturing a cell, wherein the cell produces theprotein of interest, increasing the expression or activity of a ABC50protein or a fragment thereof having protein synthesis increasingactivity and/or eIF2 binding activity, which enhances proteinproduction; culturing the cell until the protein of interestaccumulates, and isolating the protein of interest.

Another aspect provides a process for the production of a protein ofinterest comprising: culturing a cell wherein the cell comprises anexpression vector that encodes the protein of interest and an expressionvector that encodes a ABC50 protein under conditions that permitexpression of the protein of interest and the ABC50 protein; culturingthe cell until the protein of interest accumulates and isolating theprotein of interest.

In an embodiment, the process provides for the production of a proteinof interest, wherein the protein of interest is an antibody or antibodyfragment.

Another aspect relates to a method of decreasing ABC50 levels in a cellcomprising expressing an antisense agent that inhibits expression ofABC50 in the cell.

A further aspect provides a method of increasing sensitivity of a cellto ER stress agents comprising expressing an antisense agent thatinhibits expression of ABC50 in the cell.

In an embodiment, the antisense agent is a siRNA, shRNA or an antisenseoligonucleotide.

In a further embodiment, the shRNA comprises SEQ ID NO: 3 or 4.

In an embodiment, the ER stress agent is selected from EC, thapsigarginand tunicamycin.

Another aspect provides an isolated protein of interest producedaccording to a method described herein.

In an embodiment, the isolated protein produced according to a methoddescribed herein is an antibody or antibody fragment.

A further aspect provides an expression vector comprising apolynucleotide encoding an ABC50 polynucleotide and a polynucleotidecomprising a protein of interest.

A further aspect relates to a cell comprising an expression vectordescribed herein.

Yet a further aspect provides a cell comprising a heterologous ABC50gene.

Another aspect relates to a composition comprising an isolated protein,vector or cell described herein.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the disclosure will now be discussed in relation to thedrawings in which:

FIG. 1 Enhanced expression of ABC50 in Ec-resistant E2R2 cells. A:Reverse Northern analysis of genes identified by Differential Display asperformed as described in Materials and Methods. Clone 002B, identifiedin this analysis as having increased expression was sequenced and foundto be the ABC50 gene. B: Western blot of ABC50 in HL60 vs E2R2 cells.Actin expression was also evaluated to allow normalization between thetwo samples.

FIG. 2 ABC50 knockdown partially reverses resistance to Ec in E2R2cells. A: Western blot of ABC50 expression in E2R2 cells infected withvector control or ABC50 shRNA. B: Apoptosis induction by Ec in E2R2vector control and ABC50 knockdown cells. Cells were exposed to 15 μM Ecfor 2 hours followed by overnight recovery as described in Materials andMethods. The following day, cells were stained with PI and AnnexinV andanalysed by flow cytometry. AnnexinV positive, PI negative cellsrepresent early apoptotic cells, AnnexinV positive, PI positive cellsrepresent late apoptotic or necrotic cells.

FIG. 3 ABC50 knockdown alters growth rate and sensitivity to Ec in HL60cells. A: Western blot of ABC50 expression in HL60 cells infected withvector control or ABC50 shRNA. B: Cell growth kinetics of control andABC50 knocked-down cells. Values are means and standard errorsdetermined from triplicate cultures and is representative measurementfrom a series of three independent experiments. ***indicates p<0.001 at48 hours for ABC50 KD cells vs control. C: Apoptosis induction by serumwithdrawal (SW), Ec, Tg, Tu and etoposide (Eto) in HL60 vector controland ABC50 knockdown cells. Cells were exposed to 15 μM Ec for 2 hoursfollowed by overnight recovery as described in Materials and Methods.Cells were incubated overnight in the absence of serum, 200 nM Tg, 1 μMTu or 5 μM etoposide. The following day, cells were stained with PI andAnnexinV and analysed by flow cytometry. AnnexinV positive, PI negativecells represent early apoptotic cells, AnnexinV positive, PI positivecells represent late apoptotic or necrotic cells. Plotted is early andlate apoptotic cells combined. *p<0.05, **p<0.01 comparing knockdown oroverexpressing cells to their vector control.

FIG. 4 Effect of ABC50 overexpression on growth rate and sensitivity toER stress agents in HL60 cells. A: Western blot of ABC50 expression inHL60 cells infected with vector control or ABC50 OE vector. B: Cellgrowth kinetics of control and ABC50 overexpressing cells. Values aremeans and standard errors determined from triplicate cultures and isrepresentative measurement from a series of three independentexperiments. C: Apoptosis induction by serum withdrawal (SW), Ec, Tg, Tuand etoposide (Eto) in HL60 vector control and ABC50 overexpressingcells. Cells were exposed to 15 μM Ec for 2 hours followed by overnightrecovery as described in Materials and Methods. Cells were incubatedovernight in the absence of serum, 200 nM Tg, 1 μM Tu or 5 μM etoposide.The following day, cells were stained with PI and AnnexinV and analysedby flow cytometry. AnnexinV positive, PI negative cells represent earlyapoptotic cells, AnnexinV positive, PI positive cells represent lateapoptotic or necrotic cells. Plotted is early and late apoptotic cellscombined. *p<0.05, **p<0.01 comparing knockdown or overexpressing cellsto their vector control.

FIG. 5 Effect of ABC50 knockdown or overexpression on ER Ca²⁺ stores andinflux in HL60 cells. HL60 cells were loaded with the Ca²⁺-sensitive dyeIndo-1 as described in Materials and Methods. Cells were incubated (ornot) in 5 mM Ni²⁺ to non-specifically block all Ca²⁺ influx and thenexposed to thapsigargin to release ER Ca²⁺ and stimulate Ca²⁺ influx.Cytoplasmic Ca²⁺ levels were followed over time. Tg releases ER Ca²⁺ inall cases but subsequent store-operated Ca²⁺ influx is blocked in cellspre-incubated with Ni²⁺. A: HL60 cells infected with vector control orABC50 shRNA treated with Tg. B: HL60 cells infected with vector controlor ABC50 shRNA pre-incubated in Ni²⁺ to block influx, and then treatedwith Tg. C: HL60 cells infected with vector control or ABC50overexpressing virus treated with Tg. D: HL60 cells infected with vectorcontrol or ABC50 overexpressing virus pre-incubated in Ni²⁺ to blockinflux, and then treated with Tg.

FIG. 6. ABC50 knockdown or overexpression alters the ER stress response.Vector controls, knockdown (A, B) or overexpressing cells (C, D) wereexposed to Ec (15 μM), Tg (200 nM) or Tu (200 ng/ml) for 60 minutes. Thecells were collected, lysed in RIPA buffer, resolved by SDS-PAGE andanalysed with anti-sera specific for A, C: ser51-phosphorylated elF2α ortotal elF2α, B, D: BiP or actin. Numbers represent relative expressionlevels compared to control normalized to either total elF2α or actin.The blots are representative of two independent experiments.

FIG. 7 Effect of ABC50 knockdown or overexpression on ribosomal RNA andProtein content. Ribosomes were purified as described in the Materialsand Methods. A: Total ribosomal proteins obtained from 2 independentcultures and extractions were analyzed by electrophoresis on a 12%SDS-PAGE gel. The gel was then stained with Coomassie Brilliant Blue tovisualize the protein bands. B: rRNA and rProtein content as measured byabsorbance.

FIG. 8 Effect of ABC50 knockdown or overexpression on global proteinsynthesis. Cells were incubated with 15 μM Ec for 15 minutes,pulse-labelled with 3H-leucine and incorporation was measured asdescribed in Materials and Methods. A: Vector control vs ABC50knock-down. B: Vector control vs ABC50 overexpression. The values areaverages and standard errors from 4 replicates. This experiment wasrepeated six times. *p<0.05, **p<0.01 comparing knockdown oroverexpressing cells to their vector control.

FIG. 9 ABC50 overexpression increases IgG production in hybridoma GK1.5.A: GK1.5 cells were infected with empty vector control or ABC50overexpressing virus, then sorted for GFP expression. GFP expressionlevels were measured by flow cytometry. B: Control or ABC50overexpressing cells were seeded at 1×10⁶ cells/ml, cultured for 24hours, the cells were pelleted, lysed in RIPA buffer with proteaseinhibitors and cell lysates were resolved by SDS-PAGE and blotted withrabbit anti-sera specific for heavy and light chains. H: antibody heavychain, L: antibody light chain. The numbers in brackets represent theratio of band intensities (as determined by densitometry) for ABC50overexpressing vs control. The ratio is the average of three independentmeasurements. C: Cell supernatants were collected at 24 and 48 hours andIgG levels were measured by ELISA. The values are averages of twodeterminations. This experiment was repeated three times. *p<0.05comparing overexpressing cells to their vector control.

FIG. 10. Generation of Ec-resistant sp2 cells. Sp2 cells were exposed toincreasing concentrations of Econazole. Cells remaining after treatmentwere expanded and subjected to additional rounds of selection. A: cellviability for unselected (U) and selected (S) cells. Exposure toEconazole was for 2 hours in low serum medium, followed by a recoveryperiod of 24 hours in full growth medium. Cells were exposed toThapsigargin (Tg) and Tunicamycin (Tu) overnight. Cell viability wasdetermined by Trypan Blue staining of 200 cells. B: Western blot ofABC50 expression in sp2 cells selected for resistance to Ec showingincreased expression. C: Quantitation of expression normalized to actin.**p<0.01, ***p<0.001 comparing selected vs unselected.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

The term “ABC50” also known as ABCF1 refers to a member of the ATPBinding Cassette (ABC) family of proteins which lacks a transmembranedomain and includes for example human ABC50 with accession numberAF027302 (SEQ ID NO:1)¹, mouse ABC50 (e.g. SEQ ID NO:5), rat ABC50 withaccession number AF293383 (SEQ ID NO:2) (see for example,http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=85493&ordinalpos=3&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum), as well as yeast homologs yeast elongationfactor 3 (YEF3 Accession number NC_(—)001144 geneID:850951) and GCN20(Accession number NC_(—)001138, geneID:850561). Other homologs are alsocontemplated including other mammalian homologs, including but notlimited to mouse (SEQ ID NO:5; Accession number NM_(—)013854), hamster,including Chinese hamster ABC50 and insect homologs. Species homologscan be identified for example using Blast basic local alignment searchtool. In a preferred embodiment, the ABC50 is human ABC50.

The term “activity of an ABC50 protein” as used herein means a proteinsynthesis increasing activity of ABC50 protein (e.g. protein synthesisincreasing activity) which may be mediated for example by increasingtranslation initiation complex formation between elF2, GTP and/orMet-tRNA and/or by binding to elF2.

The term “Econozole” or Ec refers to an antifungal agent of theimidazole class having IUPAC name1-[2-[(4-chlorophenyl)methoxy]-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole,formula C₁₈H₁₅Cl₃N₂O and sold for example with brand names Spectazole™(US), Ecostatin™ (Canada) and Pevaryl™ (Western Europe), Endix-G™ (Asia)Ecosone™ (Thailand).

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, and chimeric antibodies as well assurface immunoglobulins. The antibody is optionally mammalian, murine,chimeric, humanized, primatized, primate, or human and can be a singlechain antibody or multichain antibody. The antibody may be fromrecombinant sources and/or produced in transgenic animals.

The term “antibody fragment” as used herein is intended to include Fab,Fab′, F(ab′)₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof and bispecific antibody fragments. Antibodies can befragmented using conventional techniques. For example, F(ab′)₂ fragmentscan be generated by treating the antibody with pepsin. The resultingF(ab′)₂ fragment can be treated to reduce disulfide bridges to produceFab′ fragments. Papain digestion can lead to the formation of Fabfragments. Fab, Fab′ and F(ab′)₂, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, bispecific antibody fragments and other fragmentscan also be synthesized by recombinant techniques.

The term “cell” as used in methods for expressing a protein of interestor increasing expression of a protein of interest refers to aneukaryotic cell, for example a yeast cell, fungi, plant cell ormammalian cell, and also includes a fused cell such as hybridoma cell.

The term “a cell” includes a single cell as well as a plurality orpopulation of cells. Contacting a cell or administering a composition toa cell includes in vivo, ex vivo and in vitro contact.

The term “protein” as used herein refers to a molecule comprised ofamino acid residues, including for example single chain polypeptides, aswell as a single chain of a multichain protein, multichain proteins suchas traditional antibodies, recombinant polypeptides including forexample fusion proteins, tagged proteins, mutant proteins and fragments,typically active fragments, of full length proteins. Protein andpolypeptide are herein used interchangeably.

The term “protein of interest” refers to a protein being produced orwhose expression is sought to be produced, by a method or processdescribed herein, and includes for example but is not limited totherapeutic proteins such as cytokines, growth factors such as insulin,coagulation factors, vaccine subunits and therapeutic enzymes, andantibodies or fragments thereof, including recombinant or naturalproteins.

The term “antibody of interest” refers to an antibody or antibodyfragment being produced or whose expression is sought to be produced, bya method or process disclosed herein. For example, the antibody ofinterest can be an antibody produced by a hybridoma whose expression issought to be increased by ABC50 overexpression.

The term “isolated protein” refers to a protein substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite activity.

In the context of a polypeptide, the term “derivative” as used hereinrefers to a polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Such derivatizedmolecules include for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included asderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline; 5hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

The term “polynucleotide” or alternatively “nucleic acid molecule” asused herein refers to a linked series of nucleoside or nucleotidemonomers consisting of naturally occurring bases, sugars and intersugar(backbone) linkages, including for example cDNA, vectors and recombinantpolynucleotides. The term also includes modified or substitutedsequences comprising non-naturally occurring monomers or portionsthereof, which function similarly. Such modified or substituted nucleicacid molecules may be preferred over naturally occurring forms becauseof properties such as enhanced cellular uptake, or increased stabilityin the presence of nucleases. The term also includes chimeric nucleicacid molecules that contain two or more chemically distinct regions. Forexample, chimeric nucleic acid molecules may contain at least one regionof modified nucleotides that confer beneficial properties (e.g.increased nuclease resistance, increased uptake into cells), or two ormore nucleic acid molecules described herein may be joined to form achimeric nucleic acid molecule. The polynucleotides may bedeoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences(RNA) and may include naturally occurring bases including adenine,guanine, cytosine, thymidine and uracil. The sequences may also containmodified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. Also, the term “nucleic acid” can be either doublestranded or single stranded, and represents the sense or antisensestrand. Further, the term “nucleic acid” includes the complementarynucleic acid sequences.

The term “isolated polynucleotide” and/or alternatively “isolatednucleic acid molecule” as used herein refers to a nucleic acidsubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated polynucleotide isalso substantially free of residues which naturally flank the nucleicacid (i.e. residues located at the 5′ and 3′ ends of the nucleic acid)from which the nucleic acid is derived.

The term “complementary” in reference to a nucleic acid as used hereinrefers to the property of a double stranded nucleic acid including DNAand RNA and DNA:RNA hybrids to base-pair according to the standardWatson-Crick complementary rules, e.g. the capacity to hybridize to aparticular nucleic acid segment under stringent conditions and/or to anucleic acid single stand that has this property e.g. is complementaryto a specific nucleic acid or portion thereof.

By “stringent hybridization conditions” it is meant that conditions areselected which promote selective hybridization between two complementarynucleic acid molecules in solution. Hybridization may occur to all or aportion of a nucleic acid sequence molecule. The hybridizing portion istypically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length.Those skilled in the art will recognize that the stability of a nucleicacid duplex, or hybrids, is determined by the Tm, which in sodiumcontaining buffers is a function of the sodium ion concentration andtemperature (Tm=81.5° C.−16.6 (Log 10 [Na+])+0.41(%(G+C)−600/I), orsimilar equation). Accordingly, the parameters in the wash conditionsthat determine hybrid stability are sodium ion concentration andtemperature. In order to identify molecules that are similar, but notidentical, to a known nucleic acid molecule a 1% mismatch may be assumedto result in about a 1° C. decrease in Tm, for example if nucleic acidmolecules are sought that have a >95% identity, the final washtemperature will be reduced by about 5° C. Based on these considerationsthose skilled in the art will be able to readily select appropriatehybridization conditions. In preferred embodiments, stringenthybridization conditions are selected. By way of example the followingconditions may be employed to achieve stringent hybridization:hybridization at 5× sodium chloride/sodium citrate (SSC)/5×Denhardt'ssolution/1.0% SDS at Tm—5° C. based on the above equation, followed by awash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridizationconditions include a washing step in 3×SSC at 42° C. It is understood,however, that equivalent stringencies may be achieved using alternativebuffers, salts and temperatures. Additional guidance regardinghybridization conditions may be found in: Current Protocols in MolecularBiology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al.,Molecular Cloning: a Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2001.

The term “control cell” as used herein refers a cell that does not haveincreased expression of an ABC50 protein or a fragment thereof havingprotein synthesis increasing activity and/or eIF2 binding activity.

The term “fragment thereof having protein synthesis increasing activity”in reference to ABC50 refers to a portion of ABC50 that retains theability to increase protein synthesis for example, by at least 5%, atleast 10% or more, for example by stimulating translation initiationcomplex formation between eIF2, GTP and/or Met-tRNA and/or binding toeIF2.

The term “fragment thereof having eIF2 binding activity” in relation toABC50 refers to an active fragment of ABC50 that binds ABC50 and retainsthe ability to increase protein synthesis.

The terms “transformed with”, “transfected with”, “transformation”“transduced” and “transfection” are intended to encompass introductionof nucleic acid (e.g. a vector) into a cell by a variety of techniquesknown in the art. The term “transformed cell” as used herein is intendedto also include cells capable of glycosylation that have beentransformed with a recombinant expression vector disclosed herein.

The term “antisense agent” as used herein means a nucleotidepolynucleotide that comprises a sequence of residues that iscomplementary to and binds a target RNA and decreases translation of itstarget RNA. For example, “antisense agents” include antisenseoligonucleotides, as well as small interfering RNAs (siRNAs) and shorthairpin RNAs (shRNAs). The nucleic acid can comprise DNA, RNA or achemical analog, that binds to the messenger RNA produced by the targetgene. Binding of the antisense agent presents translation and therebyinhibits or reduces target protein expression.

The term “siRNA” refers to a short inhibitory RNA duplex that can beused to silence gene expression of a specific gene by RNA interference(RNAi). A person skilled in the art will understand that RNAi technologyuses paired oligonucleotides. Wherein a single strand sequence isidentified by SEQ ID NO, a person skilled in the art using the rules ofbase pairing will readily determine the appropriate correspondingoligonucleotide.

The term “shRNA” refers to a short hairpin RNA. Typically shRNAs areapproximately about 50, 60 or 70 nucleotides long, or any number inbetween, for example 54 nucleotides long and can give to miRNAs. Theterm “miRNA” refers to microRNAs which are single stranded RNAs, forexample 22 nucleotides, that are processed from hairpin RNA precursors,for example about 50, 60 or 70 nucleotides long. miRNAs can inhibit geneexpression through targeting homologous mRNAs. siRNAs and shRNAsactivate a cellular degradation pathway directed at mRNAs correspondingto the siRNA or shRNA. Methods of designing specific siRNA and shRNAmolecules and administering them are described herein and known to aperson skilled in the art. For example siRNAs can comprise two 21-23nucleotide strands forming a double stranded RNA molecule, wherein onestrand is complementary to a target region in a gene of interest (e.g.comprises a sense strand homologous to the target mRNA). It is known inthe art that efficient silencing is obtained with siRNA duplex complexespaired to have a two nucleotide 3′ overhang. Adding two thymidinenucleotides is thought to add nuclease resistance. A person skilled inthe art will recognize that other nucleotides can also be added.

The term “subject”, as used herein includes all members of the animalkingdom, especially mammals, including human. The subject or patient issuitably a human.

II. Methods

ABC50 is a member of the ATP binding cassette protein family.Biochemically, ABC50 stimulates the formation of translation initiationcomplexes between eIF2, GTP and Met-tRNA implicating it in translationinitiation and control for both Cap-dependent and -independenttranslation. Econazole (Ec) is an imidazole anti-fungal that inducesendoplasmic reticulum (ER) stress in mammalian cells by promoting ERCa²⁺ depletion and sustained inhibition of protein synthesis. A previouscharacterization of HL60 cells selected for resistance to Ec found thatthe cells exhibited a phenotype of multi-drug resistance associatedspecifically with ER stress inducers. Differential Display Analysis ofthese cells identified ABC50 as a gene overexpressed in resistant cells.A similar selection process applied to sp2 cells also resulted in ERstress resistance and ABC50 overexpression. Knockdown of ABC50 in HL60cells increased sensitivity to Ec in both parental HL60 and anEc-resistant variant. ABC50 also altered sensitivity to the ER stressagents thapsigargin and tunicamycin. ABC50 knockdown increased ER Ca²⁺stores and thapsigargin-stimulated influx. Knockdown significantlysuppressed protein synthesis levels while overexpression increased them.ABC50 overexpression also increased antibody production in the hybridomaGK1.5 indicating that ABC50 overexpression is useful for theoverproduction of specific proteins. Taken together, these resultsindicate that ABC50 modulates sensitivity to Ec and other ER stressagents primarily through its effects on protein synthesis.

Accordingly, an aspect of the disclosure provides a method of producinga protein of interest comprising effecting expression of the protein ofinterest in a cell comprising an increased expression or activity of anABC50 protein or a fragment thereof having protein synthesis increasingactivity.

In an embodiment, the method comprises increasing the expression oractivity of an ABC50 protein or a fragment thereof having proteinsynthesis increasing activity in a cell; and effecting the expression ofthe protein of interest.

In another embodiment, the method comprises effecting expression of theprotein of interest in a cell comprising an increased expression oractivity of an ABC50 protein or a fragment thereof having eIF2 bindingactivity.

In yet another embodiment, the method comprises increasing theexpression or activity of an ABC50 protein or a fragment thereof havingeiF2 binding activity; and effecting the expression of the protein ofinterest.

In an embodiment, the protein of interest is a heterologous protein.

Accordingly, in an embodiment, the method comprises producing aheterologous protein of interest comprises effecting expression of theprotein of interest in a cell comprising an increased expression oractivity of an ABC50 protein or a fragment thereof having proteinsynthesis increasing activity and/or eIF2 binding activity.

In another embodiment, the method comprises producing a heterologousprotein of interest comprising increasing the expression or activity ofan ABC50 protein or a fragment thereof having protein synthesisincreasing activity and/or eIF2 binding activity; and effecting theexpression of the protein of interest.

In an embodiment, the protein of interest is produced by a cell.

In an embodiment, the protein of interest is an antibody or antibodyfragment.

Accordingly, another aspect includes a method of producing an antibody(e.g. an antibody of interest) or fragment thereof by a cell capable ofexpressing an antibody or fragment thereof comprising increasing theexpression or activity of an ABC50 protein or a fragment thereof havingprotein synthesis increasing activity and/or eIF2 binding activity inthe cell.

In an embodiment, the method comprises effecting expression of theantibody or fragment thereof in a cell comprising an increasedexpression or activity of an ABC50 protein or a fragment thereof havingprotein synthesis increasing activity and/or eIF2 binding activity.

In an embodiment, the expression or activity of ABC50 protein or afragment thereof is increased by expressing a heterologous ABC50polynucleotide encoding an ABC50 protein or a fragment thereof whereinthe ABC50 polynucleotide is operatively linked to a promoter.

Effecting expression can for example be accomplished by culturing a cellunder conditions suitable for protein expression, including for exampleculturing the cell at a growth permissive temperature, in a suitableculture medium, a sufficient time etc. that depend for example on thecell and desired expression level.

Another aspect relates to a method of increasing expression of aheterologous protein of interest by a cell expressing the protein ofinterest, comprising increasing the expression or activity of an ABC50protein or a fragment thereof having protein synthesis increasingactivity and/or eIF2 binding activity, wherein the increased expressionor activity of the ABC50 protein or fragment increases the expression ofthe heterologous protein. In an embodiment, the method comprisesintroducing a polynucleotide encoding the heterologous protein and/orintroducing a polynucleotide encoding the ABC50 protein or fragment intothe cell, for example by transfection, transduction or infection.

In an embodiment, the expression or activity of ABC50 protein or afragment thereof is increased and the expression of the protein ofinterest is effected by introducing a polynucleotide encoding the ABC50protein or a fragment thereof, and a polynucleotide encoding the proteinof interest, wherein the polynucleotides are operatively linked to oneor more promoters and optionally comprised in one or more vectors.

A further aspect relates to a method of increasing expression of anantibody or fragment thereof in a cell expressing or capable ofexpressing the antibody or fragment of interest, comprising increasingthe expression or activity of an ABC50 protein or a fragment thereofhaving protein synthesis increasing activity and/or eIF2 bindingactivity.

Cells capable of producing antibodies and/or fragments thereof may beprepared using techniques known in the art such as those described byKohler and Milstein, Nature 256, 495 (1975) and in U.S. Pat. Nos. RE32,011; 4,902,614; 4,543,439; and 4,411,993, which are incorporatedherein by reference. (See also Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Plenum Press, Kennett, McKearn, andBechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow andLane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are alsoincorporated herein by reference). Within the context of the disclosure,antibodies are understood to include monoclonal antibodies, polyclonalantibodies, antibody fragments (e.g., Fab, and F(ab′)₂) andrecombinantly produced binding partners.

For producing monoclonal antibodies the technique involveshyperimmunization of an appropriate donor with the immunogen, generallya mouse, and isolation of splenic antibody producing cells. These cellsare fused to a cell, having immortality, such as a myeloma cell, toprovide a fused cell hybrid which has immortality and secretes therequired antibody. The cells are then cultured, in bulk, and themonoclonal antibodies harvested from the culture media for use.

For producing recombinant antibodies (see generally Huston et al., 1991;Johnson and Bird, 1991; Mernaugh and Mernaugh, 1995), messenger RNAsfrom antibody producing B-lymphocytes of animals, or hybridoma arereverse-transcribed to obtain complimentary DNAs (CDNAs). Antibody cDNA,which can be full or partial length, is amplified and cloned into aphage or a plasmid. The cDNA can be a partial length of heavy and lightchain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system toobtain recombinant antibody. Antibody cDNA can also be obtained byscreening pertinent expression libraries.

As disclosed herein, ABC50 expression and/or activity can be increasedby selecting for Econozlole resistant cells. Accordingly, in anotherembodiment, the expression or activity of ABC50 protein or a fragmentthereof is increased by contacting the cell with increasingconcentrations of Econozole (Ec), and detecting increased expression oractivity of ABC50 protein. For example, Ec resistance can be induced bycontacting the cell with a sufficient concentration of Econozole (Ec) toincrease expression or activity of an ABC50 protein and selecting cellsthat maintain increased ABC50 expression and/or activity.

ABC50 expression and/or activity can be increased by introducing aheterologous ABC50 polynucleotide into a cell that is expressed.Accordingly in another embodiment, the expression or activity of ABC50protein or a fragment thereof is increased by introducing a heterologousABC50 polynucleotide encoding ABC50 protein or a fragment thereofoperatively linked to a promoter, into the cell.

In another embodiment, the cell already comprises a heterologouspolynucleotide encoding the protein of interest operatively linked to apromoter.

In a further embodiment, polynucleotides encoding ABC50 and the proteinor interest are cointroduced into a cell. Accordingly, in an embodiment,the expression or activity of ABC50 protein or a fragment thereof isincreased and the expression of the protein of interest is effected byintroducing a vector comprising a polynucleotide encoding ABC50 proteinor a fragment thereof, and a heterologous polynucleotide of the proteinof interest, wherein the polynucleotides are operatively linked to oneor more promoters. For example, expression of two polynucelotides can beachieved using an internal ribosomal entry site (IRES).

The polynucleotides may be incorporated in a known manner into anappropriate expression vector, which ensures good expression of thepolypeptides. Various constructs can be used. For example retroviralconstructs such as lentiviral constructs are useful for expressingphysiological levels of protein. Possible expression vectors include butare not limited to cosmids, plasmids, or modified viruses (e.g.replication defective retroviruses, adenoviruses and adeno-associatedviruses), so long as the vector is compatible with the host cell used.The expression vectors are “suitable for transformation of a host cell”,which means that the expression vectors contain a nucleic acid moleculeand regulatory sequences selected on the basis of the host cells to beused for expression, which is operatively linked to the nucleic acidmolecule. Operatively linked is intended to mean that the nucleic acidis linked to regulatory sequences in a manner which allows expression ofthe nucleic acid.

The disclosure therefore includes use of a recombinant expression vectorcontaining a polynucleotide molecule disclosed herein, or a fragmentthereof, and the necessary regulatory sequences for the transcriptionand translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors may also contain a selectable markergene which facilitates the selection of host cells transformed ortransfected with a recombinant molecule disclosed herein. Examples ofselectable marker genes are genes encoding a protein such as G418 andhygromycin which confer resistance to certain drugs, β-galactosidase,chloramphenicol acetyltransferase, firefly luciferase, or animmunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof the recombinant expression vectors disclosed herein and in particularto determine the effect of a mutation on expression and phenotype. Itwill be appreciated that selectable markers can be introduced on aseparate vector from the nucleic acid of interest.

Other selectable markers include for example, dihydrofolate reductase(DHFR) and glutamine synthetase (GS) for examples for use in CHO of NS0cells, respectively. Selection occurs in the absence of the metabolitese.g. glycine, hypoxanthine and thymidine for DHFR and glutamine for GS.Cells surviving selection comprise one or more copies of the transfectedplasmid in the cell's genome. Further amplification of the copy numberof the integrated DNA can be achieved by exposure of the selected cellsto increasing levels of methotrexate (MTX) or methioninen sulphoximine(MSX) respectively⁸. The recombinant expression vectors may also containgenes which encode a fusion moiety which provides increased expressionof the recombinant protein; increased solubility of the recombinantprotein; and aid in the purification of the target recombinant proteinby acting as a ligand in affinity purification. For example, aproteolytic cleavage site may be added to the target recombinant proteinto allow separation of the recombinant protein from the fusion moietysubsequent to purification of the fusion protein. Typical fusionexpression vectors include pGEX (Amrad Corp., Melbourne, Australia),pMal (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the recombinant protein.

Transcription of the protein of interest and/or ABC50 can be under thecontrol of an inducible expression system. Accordingly, in anembodiment, effecting the expression of the protein of interest and/orABC50 comprises contacting the cell with an inducer that inducesexpression of the protein of interest and/or ABC50. Examples ofinducible expression systems include the Tet-on or Tet-off inducibleexpression systems.

Recombinant expression vectors can be introduced into host cells toproduce a recombinant cell by one of many possible techniques known inthe art. For example, a polynucleotide can be introduced by transforminga cell (e.g. electroporating a prokaryotic cell), transfecting a cell(e.g. using lipofectin) or transducing a cell (e.g. using a retrovirus).Prokaryotic cells can be transformed with a polynucleotide by, forexample, electroporation or calcium-chloride mediated transformation.For example, polynucleotide can be introduced into mammalian cells viaconventional techniques such as calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran mediated transfection, lipofectin,electroporation or microinjection. Suitable methods for transforming andtransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor LaboratoryPress, 2001), and other laboratory textbooks.

In other embodiments, the cells are optionally transduced withretroviral constructs that drive expression of ABC50 and/or the proteinor antibody of interest. Methods of transducing cells are well known inthe art. Methods of transducing/infecting cells with lentiviral vectorsare also described herein.

Different ABC50 proteins can be used with the methods disclosed herein.For example, human ABC50, rat ABC50 and/or yeast ABC50 homolog can beused. Also, the ABC50 protein employed is optionally, the same speciesas the cell in which it is expressed (e.g. human ABC50, and human cell).Alternatively, the ABC50 protein employed is from a different speciesfrom the cell (e.g. human ABC50, yeast cell). Nucleic acids encodinghuman ABC50 were utilized in transfection/transduction experimentsdescribed herein and mouse Sp2 cells were treated with Ec selection. Ecselection of mouse Sp2 resulted in increased ABC50 expression asdescribed indicating that different ABC50 molecules (e.g. proteins andnucleic acids) are useful in the methods of the disclosure. Mus musculussequence is for example 88% identical and 91% similar to human ABC50according to a BLAST comparison.

In an embodiment, the ABC50 protein comprises SEQ ID NO: 1, 2 or 5; or aprotein with at least 85%, 88%, 90%, 95%, 99% or 99.5% sequence identitywith SEQ ID NO:1, 2 or 5.

In an embodiment, the ABC50 polynucleotide comprises SEQ ID NO:6, 7 or8; or a polynucleotide with at least 85%, 88%, 90%, 95%, 99% or 99.5%sequence identity with SEQ ID NO:6, 7 or 8.

In an embodiment, the method results in increased specific cellularexpression and/or production of the protein of interest in comparison toa control cell expressing the protein of interest wherein the controlcell does not have increased expression (e.g. has wildtype levels) of anABC50 protein or a fragment thereof having protein synthesis increasingactivity and/or eIF2 binding activity.

In an embodiment, the method results in increased specific cellularexpression and/or production of the antibody of interest in comparisonto a control cell expressing the antibody of interest wherein thecontrol cell does not have increased expression of an ABC50 protein or afragment thereof having protein synthesis inducing activity and/or eIF2binding activity.

In an embodiment, the increase in expression and/or production of theprotein or antibody of interest is about 5% to about 10%, about 11% toabout 20%, about 31% to about 40%, about 41% to about 50%, 51% to about60%, 61% to about 70%, 71% to about 80%, about 81% to about 90%, about91% to about 100%, about 150% to about 199%, about 200% to about 299%,about 300% to about 499%, or about 500% to about 1000%. In anembodiment, the increase is at least 5%. In another embodiment, theincrease is at least 10%.

The level of ABC50 protein and/or fragment expression and/or activity isincreased for example by an amount sufficient to increase expression ofthe protein of interest. The increase in ABC50 protein or activefragment thereof expression or activity is in an embodiment, about 5% toabout 10%, about 11% to about 20%, about 31% to about 40%, about 41% toabout 50%, 51% to about 60%, 61% to about 70%, 71% to about 80%, about81% to about 90%, about 91% to about 100%, about 150% to about 199%,about 200% to about 299%, about 300% to about 499%, or about 500% toabout 1000%. In an embodiment, the increase in ABC50 protein or activefragment thereof expression or activity is at least 10%, at least 20%,at least 30% at least 40%, at least 50%, at least 60%, at least 65% orat least 70%.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the polynucloetides and constructs that encodeproteins or antibodies of interest may be expressed in bacterial cellssuch as E. coli. Other suitable host cells can be found in Goeddel(Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. 1990).

More particularly, bacterial host cells suitable for carrying out thepresent disclosure include E. coli, B. subtilis, Salmonella typhimurium,and various species within the genus Pseudomonas, Streptomyces, andStaphylococcus, as well as many other bacterial species well known toone of ordinary skill in the art. Suitable bacterial vectors preferablycomprise a promoter which functions in the host cell, one or moreselectable phenotypic markers, and a bacterial origin of replication.Representative promoters include the β-lactamase (penicillinase) andlactose promoter system (see Chang et al. Chang et al., Nature 275:615(1978)), the trp promoter (Nichols and Yanofsky, Meth. in Enzymology101:155, 1983) and the tac promoter (Russell et al., Gene 20: 231,1982). Representative selectable markers include various antibioticresistance markers such as the kanamycin or ampicillin resistance genes.Suitable expression vectors include but are not limited tobacteriophages such as lambda derivatives or plasmids such as pBR322(see Bolivar et al. (Bolivar et al., Gene 2:9S, 1977)), the pUC plasmidspUC18, pUC19, pUC118, pUC119 (see Messing (Messing, Meth in Enzymology101:20-77, 1983) and Vieira and Messing (Vieira and Messing, Gene19:259-268 (1982)), and pNH8A, pNH16a, pNH18a, and Bluescript M13(Stratagene, La Jolla, Calif.). Typical fusion expression vectors whichmay be used are discussed above, e.g. pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.). Examples of inducible non-fusionexpression vectors include pTrc (Amann et al., Gene 69:301-315 (1988))and pET 11d (Studier et al., Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif., 60-89 (1990)).

The protein of interest can be expressed in any eukaryotic cell,including but not limited to insect cells (using baculovirus), yeastcells or mammalian cells. Yeast and fungi host cells suitable for useinclude, but are not limited to Saccharomyces cerevisiae,Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces andvarious species of the genus Aspergillus. Examples of vectors forexpression in yeast S. cerivisiae include pYepSec1 (Baldari et al., EmboJ. 6:229-234 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2(Invitrogen Corporation, San Diego, Calif.). Protocols for thetransformation of yeast and fungi are well known to those of ordinaryskill in the art (see Hinnen et al. (Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929 (1978)); Itoh et al. (Itoh et al., J. Bacteriology153:163 (1983)), and Cullen et al. (Cullen et al. Bio/Technology 5:369(1987)).

Mammalian cells suitable for use include, among others:HL60, COS (e.g.,ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No.CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1cells. Suitable expression vectors for directing expression in mammaliancells generally include a promoter (e.g., derived from viral materialsuch as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), aswell as other transcriptional and translational control sequences.Examples of mammalian expression vectors include pCDM8 (36) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the polypeptides disclosed herein may beexpressed from plant cells (see Sinkar et al., J. Biosci (Bangalore)11:47-58 (1987), which reviews the use of Agrobacterium rhizogenesvectors; see also Zambryski et al., Genetic Engineering, Principles andMethods, Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, PlenumPress, New York (1984), which describes the use of expression vectorsfor plant cells, including, among others, PAPS2022, PAPS2023, andPAPS2034).

Suitable insect cells include cells and cell lines from Bombyx,Trichoplusia or Spodotera species. Baculovirus vectors available forexpression of proteins in cultured insect cells (SF 9 cells) include thepAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and thepVL series (Luckow, V. A., and Summers, M. D., Virology 170:31-39(1989).

Alternatively, proteins and antibodies of interest may also be expressedin non-human transgenic animals such as rats, rabbits, sheep and pigs(Hammer et al. Nature 315:680-683 (1985); Palmiter et al. Science222:809-814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA82:4438-4442 (1985); Palmiter and Brinster Cell 41:343-345 (1985) andU.S. Pat. No. 4,736,866).

Accordingly, in an embodiment protein of interest is expressed by aeukaryotic cell. In an embodiment, the eukaryotic cell is selected froma yeast, plant, worm, insect, avian, fish, reptile and mammalian cell.In an embodiment, the cell is a mammalian cell. In another embodiment,the mammalian cell is a myeloma cell, a spleen cell, or a hybridoma cellproducing a specific antibody. In a further embodiment, the cell is aSp2, a NS0, a CHO, a Per.c6, a L cell. In a further embodiment, themammalian cell is a leukemia cell, such as HL-60. In the case ofincreasing expression of an antibody or fragment thereof, the ABC50protein or activity level can be increased in one or both the hybridomafusion partners and/or in the fused hybridoma cell. In anotherembodiment, the hybridoma cell is GK1.5. In a further embodiment, thecell is an Ec resistant cell. In another embodiment, the cell is an Ecresistant Sp2 cell, NS0, CHO, Per.c6, or L cell. In an embodiment, thecell is a suspension culture adapted CHO cell. In a further embodiment,the Ec resistant Sp2 cell is fused to an antibody producing spleen cell.In an embodiment, the cell is not a HEK-293 cell.

A person skilled in the art will recognize that hybridomas expressingdifferent monoclonal antibodies can be used and/or made using themethods of the disclosure.

In an embodiment, the protein of interest or antibody of interest is asecreted protein, an intracellular protein, or a membrane protein. In anembodiment, the protein of interest is a secreted protein.

Examples are provided for example in Hacker et al BioPharm Internationalincorporated herein by reference⁸. In an embodiment, the protein ofinterest is an antibody or antibody fragment or derivative thereof. Forexample, yeast cells and plant cells have been engineered to producerecombinant proteins such as recombinant monoclonal antibodies (forexample see Nature Protocols 1, 755-768 (2006); Hiatt A, Ma J, Lehner Tand Mostov K. Method for producing imunoglobulins containing protectionproteins in plants and their use 2004 U.S. Pat. No. 6,303,341; Hein M,Hiatt A and Ma J. Transgenic crops expressing assembled secretoryantibodies 2006 U.S. Pat. No. 6,995,014; Ma J K, Lehner T, Stabila P,Fux C I and Hiatt A. Assembly of monoclonal antibodies with IgG1 and IgAheavy chain domains in transgenic tobacco plants. Eur J Immunol. 1994January; 24(1):131-8; Ma J K, Hiatt A, Hein M, Vine N D, Wang F, StabilaP, van Dolleweerd C, Mostov K and Lehner T. Generation and assembly ofsecretory antibodies in plants. Science 1995, 268(5211), 716-9; Ma J K,Hikmat B Y, Wycoff K, Vine N D, Chargelegue D, Yu L, Hein M B and LehnerT. Characterization of a recombinant plant monoclonal secretory antibodyand preventive immunotherapy in humans. Nat Med. 1998, 4(5), 601-6, eachof which are herein incorporated by reference).

In an embodiment, the antibody is monoclonal, polyclonal, mammalian,murine, chimeric, humanized, primatized, primate, or human.

In an embodiment, the antibody is a fragment or derivative thereofselected from antibody immunoglobulin light chain, immunoglobulin heavychain, immunoglobulin light and heavy chains, Fab, F(ab′)2, Fc, Fc-Fcfusion proteins, Fv, single chain Fv, single domain Fv, tetravalentsingle chain Fv, disulfide-linked Fv, domain deleted, minibody, diabody,a fusion protein of one of the above fragments with another peptide orprotein or Fc-peptide fusion.

The antibody is in an embodiment, an IgG, IgM, IgA, IgD or IgE antibody.In a preferred embodiment, the antibody is an IgG antibody. In a furtherembodiment, the antibody is IgG such as IgG1, IgG2, IgG3 or IgG4.

In another embodiment, the method further comprises isolating theprotein of interest or the antibody of interest.

A variety of methods are known for isolating proteins and antibodies.The method of isolation chosen can be affected by whether the protein issecreted, membrane bound or intracellular. In an embodiment, wherein theprotein or antibody of interest is secreted, for example into a culturemedium, the method further comprising isolating the secreted protein orsecreted antibody of interest, for example from the culture supernatant.For example, the culture supernatant is collected and optionallyfractionated. In another embodiment, wherein the protein or antibody ofinterest is intracellular, the method further comprising pelletingand/or lysing the cell and isolating the intracellular protein orantibody of interest. In an embodiment, wherein the protein or antibodyof interest is membrane or surface bound, the method further comprisingsolubilizing the cell membrane and isolating the membrane protein orsurface antibody of interest. For example for antibodies, binding toantigen can be used to isolate antibodies. The most common method isprotein A columns. Other methods of purification include ammoniumsulphate precipitation, ion exchange, gel filtration and hydrophobicinteraction columns.

The disclosure also provides a process comprising the methods or aspectsdescribed herein. Accordingly, another aspect provides a process for theproduction of a protein of interest comprising: culturing a cell undersuitable culture conditions (e.g. temperature, ambient environment,culture medium, length of time etc), wherein the cell produces theprotein or antibody of interest, increasing the expression or activityof a ABC50 protein or a fragment thereof having elF2 binding activitysufficiently to enhance protein production; culturing the cell until theprotein of interest accumulates, and isolating the protein of interest.The protein of interest is an embodiment, a heterologous protein.

Another aspect provides a process for the production of a protein ofinterest comprising: culturing a cell, wherein the cell comprises anexpression vector that encodes the protein of interest and an expressionvector that encodes a ABC50 protein, under suitable culture conditions(e.g. temperature, ambient environment, culture medium etc) that permitexpression of the protein of interest and the ABC50 protein; culturingthe cell until the protein of interest accumulates and isolating theprotein of interest.

As mentioned previously, in an embodiment protein of interest is anantibody or fragment thereof.

In an embodiment, the cell is a hybridoma cell and/or a hybridoma fusionpartner.

It is also disclosed herein that decreasing ABC50 levels can be useful.Accordingly, another aspect provides a method of decreasing ABC50 levelsin a cell comprising expressing an antisense agent that inhibitsexpression of ABC50 in the cell.

For example, decreasing ABC50 levels increases sensitivity to ER stressagents. Accordingly, another aspect provides a method of increasingsensitivity of a cell to ER stress agents comprising expressing anantisense agent that inhibits expression of ABC50 in the cell.

In an embodiment, the antisense agent is a siRNA, shRNA or an antisenseoligonucleotide. In an embodiment, the antisense agent comprises SEQ IDNO:3. In another embodiment, the antisense agent comprises SEQ ID NO:4.The shRNA is in an embodiment, comprised in a lentiviral vector orvirus.

In an embodiment, the shRNA comprises SEQ ID NO: 3 or 4.

In an embodiment, the decrease in ABC50 level is about 10% to about 20%,about 21% to about 30%, about 31% to about 40%, about 41% to about 50%,51% to about 60%, 61% to about 70%, 71% to about 80%, 81% to about 90%or about 91% to about 100%.

In another embodiment, the ER stress agent is selected from EC,thapsigargin and tunicamycin.

III. Proteins and Expression Constructs

The disclosure also provides for isolated proteins produced using amethod or process described herein. Accordingly, an aspect provides anisolated protein of interest produced according to the method or processdescribed herein.

The isolated protein is in an embodiment, an antibody or antibodyfragment.

The disclosure also provides in another embodiment, an expression vectorcomprising a polynucleotide encoding an ABC50 polynucleotide andoptionally a polynucleotide comprising a protein of interest. Suitablevectors are described for example above and in the examples below.

In an embodiment, the vector comprises a polynucleotide encoding anABC50 polynucleotide and optionally a polynucleotide encoding a proteinof interest, wherein the polynucleotide(s) is/are operably linked to oneor more promoters. In an embodiment, the vector is a retroviral vector,optionally a lentiviral vector.

IV. Cells

Another aspect provides a recombinant and/or isolated cell. In anembodiment, the recombinant cell comprises a vector described herein. Inanother embodiment, the recombinant cell comprises a heterologous ABC50gene. In yet a further embodiment, the cell comprises an EC resistantcell comprising increased ABC50 expression or activity.

In and embodiment, the cell comprises a heterologous ABC50polynucleotide operably linked to a promoter or an Ec resistant cellwherein the Ec resistant cell has increased ABC50 protein levels oractivity compared to a non-Ec resistant control cell, wherein the cellis suitable and/or adapted for expression of a protein of interest. Forexample, a hybridoma fusion partner cell is such a suitable cell as ahybridoma fusion partner cell expressing the increased ABC50 is usefulfor fusing with any antibody cell to produce a hybridoma with increasedantibody production compared to a hybridoma cell not comprisingincreased ABC50 expression. As another example, any eukaryotic cell thatis transfectable, transduceable or infectable and that is useful forexpressing proteins, for example in large amounts, is also a suitablecell.

In an embodiment, the EC resistant cell is an Ec resistant SP2 cell, CHOcell, NS0 cell, a Per.c6 or L cell.

Suitable host cells are described above. In an embodiment, the cell isselected from a yeast, plant, worm, insect, avian, fish, reptile,mammalian, hybridoma, a myeloma cell or a spleen cell.

A further aspect provides a system for increasing expression of aprotein of interest, the system comprising a cell comprising increasedexpression or activity of ABC50. For example, the cell can be a frozencell or a lyophilized cell. In an embodiment the system furthercomprises an expression vector in which can be introduced apolynucleotide encoding a protein of interest. In an embodiment, theABC50 expression or activity increase results from introduction of aheterologous polynucleotide encoding ACB50. In another embodiment, theABC50 expression or activity increase results from selection with Ec. Ina further embodiment, the system comprises Ec such as in a form suitablefor administration to a cell to maintain selective pressure, for exampleas a stock solution in DMSO for administering to cells at aconcentration of for example 5, 10, 15, 20 or 25 microM.

V. Compositions

In another aspect, the isolated protein, vector or recombinant cell iscomprised in a composition. In yet a further embodiment, the compositioncomprises a polynucleotide comprising SEQ ID NO:3. In anotherembodiment, the composition comprises a polynucleotide comprising SEQ IDNO:4. In a further embodiment, the composition comprises a carrier. Inanother embodiment, the carrier is a pharmaceutically acceptablecarrier. In a further aspect, the composition is for decreasing thelevel of ABC50.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

Recently, the inventor showed that transformation by the c-myc oncogenesensitizes cells to Ec by enhancing ROS generation at the mitochondria⁹providing at least one mechanism by which cancer cells exhibitsensitivity to Ec.

Previously, the inventor generated and characterized variants of HL60cells that were resistant to Ec¹⁰. Although selected for resistance toEc, the cells also displayed resistance to other ER stress agentsincluding thapsigargin, tunicamycin, DTT and cycloheximide, thusdefining a novel phenotype of multi-drug resistance associated with ERstress. Resistance was found to be associated with increasedstore-operated Ca²⁺ influx capability and sustained protein synthesisafter exposure to Ec. Microarray analysis of a resistant clone revealedincreased expression of ribosomal protein genes. Biochemical analysisshowed that this increased gene expression was associated with increasedribosomal content. Ribosome inactivating toxins partially reversedresistance to ER stress suggesting that the increased ribosomal contentand function contributed to resistance.

To further identify genes associated with resistance and sensitivity toEc, the inventor performed differential display analysis¹¹ comparing theEc-resistant cell line E2R2 with parental HL60 cells. This analysisidentified ABC50 as a gene overexpressed in Ec-resistant cells. ABC50contributes to Ec-resistance.

Results Differential Display of Ec-Resistant Vs Sensitive HL60 Cells.

In order to identify additional genes associated with Ec resistance,Differential Display analysis was performed¹¹ comparing Ec-resistantE2R2 cells with parental HL60 cells. This analysis identifiedapproximately 200 gene fragments that appeared to be overexpressed inE2R2 cells compared to Wild Type. These gene fragments were cloned andReverse Northern analysis was employed to confirm differentialexpression. 50 of the 200 genes had expression levels above thedetection limit of the Reverse Northern. Of the 50, 15 genes wereconfirmed to be differentially expressed. Sequence analysis identifiedthese genes as follows: Two of the 15 encoded ribosomal protein genes,three encoded Alu-containing sequences, two were mitochondrial genes andone gene encoded the integrin CD11a. Two genes were identified that areclassified as TNFα inducible. These were HLA gene (Bw-62), and ABC50(NM_(—)001090; aka ABCF1), a member of the ATP binding cassette family(FIG. 1A). Two additional genes of unknown function with no knownhomology or similarity to any other gene (AC114546, AC012358) wereidentified. One codes for hypothetical protein FLJ12363 (XP_(—)043979)with no known function. The final gene identified in this screen waspolyubiquitin C (AB009010). The protein and nucleic acid sequence of theaformentioned genes referred to by accession number, are hereinspecifically incorporated by reference.

ABC50 Protein Levels in E2R2 Cells.

ABC50 was investigated. It was first confirmed that ABC50 wasoverexpressed in E2R2 cells. As shown in FIG. 1B, increased levels ofABC50 protein were detected in E2R2 cells compared to HL60 cells.Densitometric analysis of Western blots indicated a 65% increasedexpression (relative to actin) of ABC50 in E2R2 compared to HL60 cells.

ABC50 Knockdown (KD) in E2R2 Cells.

The association of ABC50 with the Ec-resistance phenotype of E2R2 cellswas further investigated by knocking down its expression in these cells.The cells were infected with a lentiviral vector expressing shRNAspecific for ABC50 and sorted based on GFP expression. As shown in FIG.2A, ABC50 knockdown was successful in these cells (36% relative decreasecompared to vector control). Furthermore, as shown in FIG. 2B, ABC50knockdown in E2R2 cells partially reversed their resistance to Ec (21.4%combined early and late apoptosis compared to 7.6% combined early andlate apoptosis in the control cells), consistent with a role for ABC50in the Ec resistance phenotype.

ABC50 Knockdown in HL60 Cells Increases Sensitivity to ER Stress Agents.

To investigate further the consequences of manipulating ABC50 levels incells, parental HL60 cells were infected with the lentiviral vectorexpressing shRNA specific for ABC50 and sorted infected cells based onGFP expression. As shown in FIG. 3A, the shRNA knocked down expressionof ABC50 by 89% compared to vector control. Light microscopicobservation revealed that the cells had no obvious morphologicaldifferences. It was also found that the knocked-down cells grew at arate that was not significantly different from the control cells (FIG.3B).

The effect of ABC50 knockdown on sensitivity to Ec and otherapoptosis-inducing agents was next investigated. Tg is a classic inducerof ER stress and HL60 cells selected for resistance to Ec were alsofound to be resistant to Tg. Sensitivity to Tunicamycin (Tu), aninhibitor of protein glycosylation and another classic inducer of ERstress was also tested. As shown in FIG. 3C, ABC50 knockdownsignificantly increased the sensitivity of HL60 cells to Ec, Tg and Tu.In contrast, ABC50 KD did not affect sensitivity to serum withdrawal orthe topoisomerase inhibitor etoposide. This observation suggests thatABC50 knockdown specifically increases sensitivity to ER stress-inducingagents.

ABC50 Overexpression in HL60 Cells Decreases Sensitivity to ER StressAgents.

The observation of increased ABC50 expression in the Ec-resistant E2R2cells suggested that overexpression of the gene might promoteresistance. To investigate this possibility, HL60 cells were infectedwith a lentiviral vector expressing the full ABC50 coding sequence,infected cells were sorted as above using the GFP marker, and the cellphenotype was analysed. As shown in FIG. 4A, infection with the ABC50lentiviral vector significantly increased expression of the protein (42%relative increase compared to vector control). Cell growth propertieswere measured and it was found that the ABC50 overexpressing cells hadno significant differences in growth kinetics compared to control HL60cells infected with vector alone (FIG. 4B). However as shown in FIG. 4C,ABC50 overexpressing cells displayed decreased sensitivity to the ERstress agents Ec, Tg and Tu whereas their sensitivity to serumwithdrawal or etoposide was unchanged compared to control cells. Takentogether, these results demonstrate that ABC50 expression levelsspecifically affect sensitivity to ER stress.

ER Ca²⁺ Content and Influx in ABC50 Knockdown and Overexpressing Cells.

It was previously demonstrated that the Ec resistance phenotype of E2R2cells was associated with altered Ca²⁺ physiology. Specifically, E2R2cells displayed unchanged ER Ca²⁺ store content, but increased Ca²⁺influx in response to ER Ca²⁺ store depletion by the ATPase ER Ca²⁺ pumpinhibitor thapsigargin¹⁰. To investigate the effect of altered ABC50expression on Ca²⁺ physiology, ER Ca²⁺ content and influx was measuredin ABC50 knockdown and overexpressing cells. As shown in FIG. 5, nodifferences in either ER Ca²⁺ content (FIG. 5B, D) or Tg-stimulated Ca²⁺influx (FIG. 5A, C) were observed in ABC50 KD or overexpressing cells.These observations indicate that ABC50 does not directly affect Ca²⁺physiology in HL60 cells.

ER Stress Response in ABC50 Knockdown or Overexpressing Cells.

Ec, Tg and Tu are all potent inducers of ER stress. To compare the ERstress response of cells with altered ABC50 expression, cells weretreated for 60 minutes with the ER stress agents and levels ofphosphorylated eIF2α and the chaperone BiP, two classic indicators of ERstress, were determined by Western blot. As shown in FIG. 6A, increasedlevels of phosphorylated eIF2α were observed in treated ABC50 knockdowncells compared to vector control. Ec and Tg were particularly effectiveat inducing increased levels of eIF2α. Induction of BiP expression by ERstress agents was not affected by ABC50 knockdown (FIG. 6B) althoughbasal levels were slightly increased compared to control. In contrast,ABC50 overexpressing cells displayed reduced levels of phosphorylatedeIF2α when exposed to Ec, Tg and Tu (FIG. 6C). BiP expression was littlechanged in response to the ER stress agents compared to control (FIG.6D) with no observed difference in background expression. Takentogether, the divergence of response between eIF2α phosphorylation andBiP induction suggests that the effect of ABC50 is specific for theeIF2α response.

Ribosomal Content and Protein Synthesis in ABC50 Knockdown orOverexpressing Cells.

Two major biochemical differences observed previously in Ec-resistantcells were increased ribosomal content and sustained protein synthesisin response to Ec¹⁰. As shown in FIGS. 7A and B, a trend was observedtoward decreased ribosomal RNA and Protein in ABC50 knockdown cells andincreased levels in ABC50 overexpressing cells. To test the effect ofaltered ABC50 expression on protein synthesis, ABC50 knock-down oroverexpressing cells were exposed to Ec and global protein synthesisrates were measured. As shown in FIG. 8A, exposure of control cells toEc resulted in a significant decrease in protein synthesis levels.Interestingly, ABC50 knock down cells displayed a lower base rate ofprotein synthesis compared to control. Addition of Ec reduced proteinsynthesis rates even further. In contrast, ABC50 overexpressing cellsdisplayed a slightly higher level of protein synthesis compared tocontrol cells and this level was significantly less reduced afterexposure to Ec (FIG. 8B). Taken together, these observations indicatethat altered ABC50 expression affects ribosomal content, basal proteinsynthesis and modifies the cellular response to Ec on protein synthesis.

Enhanced IgG Production in ABC50 Overexpressing Hybridoma Cells.

The observation that ABC50 expression influenced global proteinsynthesis levels suggested that it might also affect expression ofindividual proteins. This property might be of utility in enhancingproduction of useful proteins, particularly in cells expressing highamounts of specific proteins such as hybridomas. To test thispossibility, hybridoma cell line GK1.5 was infected with theABC50-expressing lentivirus, infected cells were sorted using GFPexpression as a marker of infection (FIG. 9A) and antibody productionwas measured by Western blotting and ELISA. As shown in FIG. 9B, GK1.5cells infected with the ABC50 expressing virus produced significantlymore antibody heavy and light chains compared to vector control. ELISAanalysis of antibody concentrations secreted into the supernatantindicated that antibody production was 44% higher at 48 h in ABC50overexpressing cells compared to control cultures. (FIG. 9C). Thisresult suggests that ABC50 is useful in boosting protein expression ofspecific gene products like antibody heavy and light chains.

Selection for Ec Resistance in Sp2 Cells Results in Multidrug Resistanceand Increased ABC50 Expression.

Sp2 cells are commonly used as fusion partners for creating hybridomas.The ability to generate sp2 cells that are generally resistant to ERstress and overexpress ABC50 would therefore be of use in the process ofhybridoma generation. To this end, sp2 cells were exposed to increasingconcentrations of Ec as described above for HL60 cells. Theirsensitivity to ER stress agents was then characterized. As shown in FIG.10 a, Ec-resistant sp2 cells were also found to be relatively resistantto the other ER stress agents Tg and Tu. Furthermore, expressionanalysis (FIGS. 10B,C) indicates that ABC50 is also overexpressed inthese cells. These observations therefore indicate that Ec selection isuseful for selecting ER stress resistant and ABC50 overexpressing cells.

Discussion

Ec induces ER stress and cell death through the sustained depletion ofER Ca²⁺ stores. This is caused by blocking Ca²⁺ influx at the plasmamembrane and stimulating ER Ca²⁺ release through ROS generation at themitochondria. One consequence of this Ca²⁺ depletion effect is profoundinhibition of protein synthesis. The generation and characterization ofEc-resistant mutants further supported the importance of Ca²⁺ depletionand protein synthesis inhibition by demonstrating increased influx andincreased ribosomal content and function in resistant cells. A role forthe protein ABC50 in Ec resistance is disclosed. ABC50 is hereinidentified as an overexpressed gene in Ec-resistant E2R2 cells. Westernblot analysis demonstrated that protein levels were increased by 65%compared to WT cells. Sp2 cells similarly selected for Ec resistancewere also observed to be multi-drug resistant and to overexpress ABC50.Knockdown of ABC50 in both HL60 and E2R2 cells increased sensitivity toEc indicating that ABC50 contributes to resistance. ABC50 was also foundto modulate sensitivity to Tg and Tu, other ER stress agents but notserum withdrawal or etoposide. ABC50 knockdown had no effect on ER Ca²⁺content and influx, but reduced ribosomal content and protein synthesisin knock-down cells and increased ribosomal content and proteinsynthesis in HL60 cells overexpressing the protein. Taken together,these results indicate that ABC50 affects sensitivity to Ec and other ERstress agents, likely through its effects on protein synthesis.

It is of interest to contrast the effect of ABC50 knock-down with ABC50overexpression. While the knock-down significantly increased ER stressindicators eIF2α and BiP, decreased protein synthesis and increasedsensitivity to Ec, overexpression only slightly relieved ER stressindicators and increased protein synthesis and had only a modest effecton Ec sensitivity. The observation of effects on protein synthesisthrough ABC50 overexpression differs from the recent work of Paytubi etal. who observed a lack of effect on protein synthesis afteroverexpressing ABC50 in HEK293 cells³. Without wishing to be bound bytheory, these observations indicate that a reduction in its proteinlevel may make ABC50 rate-limiting for protein synthesis while themodest effect from overexpression indicates that ABC50 is not normallyrate-limiting. As well, while ABC50 overexpression did partially preventfull inhibition of protein synthesis by Ec, this effect was insufficientto provide significant protection from Ec-induced apoptosis. Thisobservation may indicate that full resistance to Ec requires bothaltered Ca²⁺ influx as well as increased protein synthesis.

Manipulating ABC50 expression levels was shown to also alter sensitivityto the classic ER stress inducers Tg and Tu. Tg, like Ec, depletes theER of Ca²⁺. However unlike Ec, which blocks Ca²⁺ influx, ER depletion byTg overstimulates influx resulting in very high cytoplasmic Ca²⁺ levels(FIGS. 5A,C). This Ca²⁺ overload response likely contributessignificantly to Tg-induced apoptosis, as documented previously in mastcells⁴. Therefore the partial effect of ABC50 knockdown on Tgsensitivity may reflect the relative importance of Ca²⁺ overloadcompared to ER stress in Tg toxicity. Tu is a glycosylation inhibitorand induces ER stress through the Unfolded Protein StressResponse^(12,13). Since one consequence of ER stress induction issuppression of protein synthesis, it is possible that ABC50 knockdownpromotes Tu toxicity through a combined effect on protein synthesis.Nevertheless, the fact that ABC50 overexpression partially protectscells from Ec, Tg and Tu indicates that its overexpression contributedto the multi-drug resistance phenotype of E2R2 cells.

As shown in FIG. 6, increased phosphorylation of eIF2α was observed inresponse to ER stress when ABC50 was knocked down, and decreased levelswhen ABC50 was overexpressed. Tyzack et al.² previously commented thatthey did not observe any effect of ABC50 on eIF2α phosphorylation by RNAPK in vitro. Without wishing to be bound to therory, the observationthat eIF2 phosphorylation is modulated by ABC50 may reflect the uniqueenvironment of ER stressed cells. Alternatively, the effects of ABC50 oneIF2a phosphorylation may be an indirect effect associated with alteredcellular stress due to insufficient (or excess) ABC50. The fact that BiPinduction is little changed when ABC50 expression is altered arguesagainst a general effect on ER stress.

Ribosomal biogenesis is tightly regulated during growth through the mTORpathway^(14,15). Cellular stress can also influence ribosome biogenesisthrough both mTOR and JNK-mediated phosphorylation of the TIF-IAtranscription factor¹⁶, resulting in inhibition of rDNA transcription.The observation of reduced and increased ribosomal content in ABC50 KDor overexpressing cells respectively is unlikely to reflect growthconditions, since growth rate in both cases appeared to be similar toWT. It is possible that altered ribosomal content reflects differencesin basal stress levels, as indicated by increased BiP and phospho-eIF2αlevels in ABC50KD cells.

Although modest, the increased level of protein synthesis due to ABC50overexpression translated into a significant increase in antibodyproduction by the hybridoma GK1.5. Therefore, increasing ABC50expression is useful for boosting expression of specific proteins ofinterest such as antibody heavy and light chains. Interestingly, Ota etal.¹⁷ recently identified a genetic linkage between the ABC50 gene locusand increased susceptibility to auoimmune pancreatitis. Since thephenotype of these patients includes increased serum titers of IgG₄, itis possible that ABC50 polymorphisms may contribute to this disease byenhancing antibody production.

ABC50 contributes significantly to Ec resistance. Its mechanism ofaction appears to be primarily through its modulation of proteinsynthesis.

Materials and Methods Cells and Cell Culture

Human HL60 promyelocytic leukemia cells, their E2R2 derivative and GK1.5hybridoma cells¹⁸ were cultured in RPMI 1640 medium supplemented with10% FBS and antibiotics.

Growth Curves

Cells were grown in duplicate cultures at the initial concentration of0.4×10⁶ in RPMI containing 10% FCS. Cells were collected and counted atthe 24, 48 and 72 hour time intervals.

Apoptosis

To measure apoptosis induced by Econazole (Ec; Sigma-Aldrich, St. Louis,Mo.), cells were treated with Ec in RPMI containing 1% FBS for 2 hoursat 37° C. then further incubated overnight in RPMI containing 10% FBS.Apoptosis induced by thapsigargin (Tg; Sigma-Aldrich) or Tunicamycin(Tu; Sigma-Aldrich) was determined after overnight incubation in RPMIcontaining 1% FBS. The cells were washed with PBS and stained withAnnexin V-cy5 Apoptosis Detection kit (Biovision. Inc., Mountain View,Calif.)/PI and analysed by flow cytometry.

Differential Display

Differential Display¹¹ comparing mRNA from HL60 vs Ec-resistant E2R2cells was performed using the Delta Differential Display Kit fromClontech. All procedures were performed according to the manufacturer'sinstructions and involved using pairwise combinations of 10 Arbitraryprimers with 9 Oligo dT primers. Differentially expressed bands wereexcised from the gel, re-amplified, TA-cloned and sequenced.

Reverse Northern Analysis

3 μg of plasmid DNA from each sample was boiled, rapidly placed on ice,then dotted through a dot blot manifold onto duplicate pre-soaked nylonmembranes. The membranes were U.V. cross-linked, incubated inpre-hybridization solution (5×SSC, 5×Denhardt's solution, 50 mM PBS (pH7.0), 0.2% SDS, 500 μg/ml salmon sperm DNA, 50% formamide). Themembranes were hybridized in hybridization solution (5×SSC, 5×Denhardt'ssolution, 50% formamide) containing 6.5×10⁷ cpm of ³²PdCTP-labelledreverse-transcribed cDNA probe from either HL60 or E2R2 total RNA. Theblots were hybridized overnight, washed in 2×SSC and then 2×SSC, 0.1%SDS until background radiation was reduced. The blots were exposed tox-ray film for visualization.

Construction of Lentivirus Vectors

The empty lentivirus vector pLEN (H1GFP), in which the H1 promoterdrives expression of shRNA sequences was a gift from Dr. John Dick(University Health Network, Toronto, Canada). The sequences of theoligos used to knock down ABC50 expression were:5′TAAGCTGTCATCTGGCTTAATAAGGATCCTTATTAAGCCAGATGA CAGCTTTTT3′ (SEQ IDNO:3) and 5′CTAGAAAAAGCTGTCATCTGGCTTAATAAGGATCCTTATTAAGCCAGATGACAGCTTAAT3′ (SEQ ID NO:4). Each pair of oligos were mixed and annealedby incubating at 95° C. for 5 min and cooling slowly. The annealedmixture was ligated into pLEN vector that had been digested with Pacland Xbal.

Construction of Lentivirus Over-Expressing ABC50

The ABC50 clone 7 (obtained from Dr. A. Beaulieu, University of Laval,Quebec, Canada, missing 4 nt from the 5′ end) (GenBank Accession number:AF027302; gi: 2522533) was used as the template for cloning the ABC50structural gene by PCR amplification. To add the 4 nt at the 5′, twoprimers were used: Forward: 5′-AT CCCGGG ATGC CGA AGG CGC CCA AGC AGCAGC-3′ (SEQ ID NO:9)(contains Xmal site); Reverse: 5′-AT CTCGAG TCAC TCTCGG GGC CGG CTG ACC-3′ (SEQ ID NO:10) (contains Xhol site). Theamplified ABC50 structural gene was first cloned into pCR4Blunt-TOPOvector (Invitrogen) then subcloned into the pCE lentivirus expressionvector (Dr. John Dick, U H N, Toronto) that has been digested with Xmaland Xhol. The whole ABC50 gene was sequenced to confirm the lack ofmutations.

Generation of the Infective Lentivirus

Lentivirus vectors harboring human ABC50 shRNA or the ABC50 structuralgene were produced by transient transfection into 293T cells aspreviously described¹⁹. Briefly, the backbone plasmid vector construct(10 μg) was mixed with the accessory plasmids VSVG (3.5 μg), pRRE (6.5μg) and pREV (2.5 μg) and transfected into 293T cells with the CalphosMammalian Transfection Kit (Clontech, Mountain View, Calif.). The cellsupernatant was replaced with 4 ml fresh Iscove MEM (10% FCS) at 24hours and virus was harvested at 48 hours after the plasmidtransfection.

Lentiviral Infection

A total of 0.1×10⁶ HL60 cells were infected with 2 ml lentivirus culturesupernatant (˜2×10⁶ virus particles) in the presence of 8 μg/mlpolybrene (Sigma-Aldrich, St. Louis, Mo.) for 4 days. Up to 94% of cellswere positive for GFP expression. GFP positive cells were sorted byfluorecence activated cell sorting and grown in RPMI (10% FBS) forfurther analysis.

Western Blot

Cells were washed with PBS and lysed with Triple lysis buffer (50 mMTris pH7.0, 150 mM NaCl, 0.1% SDS, 1% NP-40 and 0.5% DOC). Proteinaseinhibitor (Boehringer) was added to 10 ml lysis buffer before use.Protein concentration was determined with the Pierce BCA kit. 20 μg oftotal protein was loaded onto 10% SDS-PAGE, transferred onto filters andblotted with rabbit anti-human ABC50 polyconal serum (kind gift from Dr.C. Proud, Vancouver, Canada). eIF2α and its phosphorylated form (Ser51)were detected with rabbit polyclonal antibodies from Cell Signalling(Danvers, Mass.). Mouse anti-BiP/GRP78 antibodies were obtained from BDBiosciences (San Jose, Calif.) Anti-actin (pan Ab-5, Clone ACTN05)(Labvision/Neomarker, Fremont, Calif.) was used as a loading control.

Ca²⁺ Measurement

[Ca²⁺]_(c) measurements were performed by flow cytometry. Cells (5×10⁵cells/ml) were serum-deprived for ˜2 hours in Tyrode's buffer [HEPES (10mM), NaCl (100 mM), KCl (5 mM), CaCl₂ (1.4 mM), MgCl₂ (1 mM), glucose(5.6 mM), BSA (0.05%)]. Cells were then incubated in Indo-1 loadingbuffer (30 min, 37° C.; 5 μM Indo-1AM, 0.03% pluronic F-127 in Tyrode'sbuffer), washed (2 times) and incubated at room temperature (greaterthan 15 min) to allow for the complete removal and/or conversion ofIndo-1AM to Ca²⁺-sensitive Indo-1. Measurements were performed using alaser tuned to 338 nm while monitoring emissions at 405 nm and 450 nm.The concentration of intracellular free Ca²⁺ was calculated according tothe following formula²⁰:

[Ca²⁺]_(i)=K_(d)×(F_(min)/F_(max))×(R−R_(min))/(R_(max)−R),

where R is the ratio of the fluorescence intensities measured at 405 nmand 450 nm during the experiments and F is the fluorescence intensitymeasured at 450 nm. R_(min), R_(max), F_(min) and F_(max) weredetermined from in situ calibration of unlysed cells using 4 μMionomycin in the absence (R_(min) and F_(min); 10 mM EGTA) and presenceof (R_(max) and F_(max)) of Ca²⁺. K_(d) (250 nM) is the dissociationconstant for Indo-1 at 37° C. R_(min), R_(max), F_(min) and F_(max)varied depending upon settings and were determined at the beginning ofeach experimental procedure.

Protein Synthesis

Cells (2×10⁵/sample) were collected, washed with PBS and thenre-suspended in RPMI supplemented with fatty acid-free bovine serumalbumin (BSA; 0.05%; Sigma). Cells were treated with Ec (0, 15 μM) for15 min. After centrifugation (2,500 rpm; 5 min), cells werepulse-labeled with [³H]-leucine (50 μCi/ml) for 10 min (37° C.; 5% CO₂)in leucine-free RPMI. After two washes in RPMI, pellets were lysed withTriton X-100 (0.5% in PBS) followed by trichloroacetic acid (TCA, 10%w/v; 4° C.). Samples were washed in TCA (5% w/v), and the proteinpellets were re-suspended in microscintillant (Packard, Conn., USA) andmeasured using a microplate scintillation counter (Packard).

Ribosomal Purification

5×10⁷ HL-60 cells growing in log phase were collected, washed with coldPBS, and fractionated according to the method described by Greco andMadjar²¹. The ribosomal fraction was isolated through centrifugation ofpost mitochondrial supernatants on top of a 1 M sucrose cushion at245,000×g to pellet the ribosomes. The ribosome pellets were resuspendedin 300 μl of RIPA buffer and disrupted by incubation in 60 mM EDTA onice for 30 min. The concentration of the total ribosomal protein wascalculated based on the absorbance of the samples (A280). Ribosomal RNAswere extracted with TRIzol and the concentration was measured by aspectrophotometer at A260.

IgG Measurements

IgG levels produced by the rat hybridoma GK1.5 (ATCC no. TIB-207) weremeasured by Western blotting and ELISA. For Western blotting, cells wereplated at a concentration of 1×10⁶ cells/ml in growth medium for 24hours. The cells were then collected, counted, pelleted and cell lysateswere prepared in RIPA buffer with protease inhibitors (Sigma). Lysatesand cell supernatants were resolved on 10% SDS-PAGE and transferred toPVDF membranes. Antibody heavy and light chains were detected withHRP-conjugated rabbit anti-rat IgG (H+L) (Zymed; San Franciso, Calif.).For ELISA measurements, Goat anti-rat IgG or normal control IgG fromGoat serum (Sigma) were diluted to 5 μg/ml in coating buffer (50 mMTris, 150 mM NaCl, pH9.5), placed into a 96 well ELISA plate in 50 μlvolume and incubated for 40 min at room temperature. The plate waswashed for 8 times with distilled water and incubated with 50 μl of PBScontaining 3% FBS for additional 40 min at room temperature. Emptyvector and ABC50 over-expressed lentivirus transfected GK1.5 hybridomacells were grown in Iscove's Modified Dulbecco's Medium (IMDM)containing 10% FBS. Cell culture supernatant was collected and dilutedin same media and 50 μl diluted samples were added into the 96-wellplate. Normal rat IgG from rat serum (Sigma) was used for determiningthe standard curve. After incubating for 2 hours at room temperature,the wells were washed 8 times with distilled water. HRP conjugated Goatanti-rat IgG (Sigma) was diluted 1:2000 in IMDM and 50 μl reagent wasadded, incubated for another 40 min. and washed as described above. 100μl of substrate 3,3,5,5-Tetramethylbenzidine (TMB) (Sigma) was added andthe reaction was stopped with 0.5 M H₂SO₄ when a yellow color developed(5 to 10 min). The plate was read at 450 nM with an ELISA reader.

Statistical Analysis

Where indicated, statistical significance was determined using theStudent's t-test. p<0.05 (*), p<0.01 (**) and p<0.001 (***) were asindicated.

Example 2 Methods of Producing a Protein of Interest

There are various methods to effect expression of a protein of interest.For example, a cell expressing a protein of interest, endogenous orheterologous can be transfected/transduced with an expression vector orinfected with a virus to introduce a ABC50 polynucleotide encoding aABC50 protein or fragment having protein synthesis increasing activity.For example, a method can comprise:

Transfect/transduce cells expressing a protein of interest with theABC50 expression vector. Cells could be selected using a drug-resistancemarker, or by expression of a co-transduced marker like GFP.

Alternatively, cells overexpressing ABC50 can be made to express theprotein of interest. For example, a method can comprise the following:

Overexpress ABC50 in cells, then transfect them with the recombinantprotein of interest.

In a further alternative, the protein of interest and ABC50 protein orfragment, can be coexpressed for example by transfect/transduce cellswith ABC50 and the protein of interest together.

Further Ec selection can be used to increase ABC50 levels in a cellexpressing a protein of interest and/or in a cell into which apolynucleotide encoding a protein of interest is introduced. For examplesuch a method could comprise: select cells that are resistant to Ec andthen use them as recipients for further transfection with a protein ofinterest.

Table: Sequences 1. Examples of Human ABC50 Molecules

A human ABC50 amino acid sequence is provided in SEQ ID NO:1A human ABC50 nucleotide sequence is provided in SEQ ID NO:6

2. Examples of Rat ABC50 Molecules

A rat ABC50 amino acid sequence is provided in SEQ ID NO:2A rat ABC50 nucleotide sequence is provided in SEQ ID NO:7

3. Examples of Mouse ABC50 Molecules

A mouse ABC50 amino acid sequence is provided in SEQ ID NO: 5A mouse ABC50 nucleotide sequence is provided in SEQ ID NO: 8

4. Examples of Antisense Agents

(SEQ ID NO: 3) 5′TAAGCTGTCATCTGGCTTAATAAGGATCCTTATTAAGCCAGATGACAGCTTTTT3′ (SEQ ID NO: 4)5′CTAGAAAAAGCTGTCATCTGGCTTAATAAGGATCCTTATTAAGCCAGA TGACAGCTTAAT3′

5. Examples of Primers for Cloning ABC50

(SEQ ID NO: 9) 5′-AT CCCGGG ATGC CGA AGG CGC CCA AGC AGC AGC-3′(contains XmaI site); (SEQ ID NO: 10)5′-AT CTCGAG TCAC TCT CGG GGC CGG CTG ACC-3′ (contains XhoI site)

While the present disclosure has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the disclosure is not limited to the disclosed examples.To the contrary, the disclosure is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

-   1. Richard M, Drouin R, Beaulieu A D. ABC50, a novel human    ATP-binding cassette protein found in tumor necrosis    factor-alpha-stimulated synoviocytes. Genomics. 1998; 53:137.-   2. Tyzack J K, Wang X, Belsham G J, Proud C G. ABC50 interacts with    eukaryotic initiation factor 2 and associates with the ribosome in    an ATP-dependent manner. J Biol Chem. 2000; 275:34131.-   3. Paytubi S, Morrice N A, Boudeau J, Proud CG. The N-terminal    region of ABC50 interacts with eukaryotic initiation factor eIF2 and    is a target for regulatory phosphorylation by CK2. Biochem J. 2008;    409:223.-   4. Soboloff J, Berger S A. Sustained ER Ca2+ Depletion Suppresses    Protein Synthesis and Induces Activation-enhanced Cell Death in Mast    Cells. J Biol Chem. 2002; 277:13812.-   5. Zhang Y, Soboloff J, Zhu Z, Berger SA. Inhibition of Ca2+ influx    is required for mitochondrial reactive oxygen species-induced    endoplasmic reticulum Ca2+ depletion and cell death in leukemia    cells. Mol Pharmacol. 2006; 70:1424.-   6. Soboloff J, Zhang Y, Minden M, Berger S. Sensitivity of myeloid    leukemia cells to calcium influx blockade. Application to bone    marrow purging. Exp Hematol. 2002; 30:1219.-   7. Zhang Y, Crump M, Berger S A. Purging of contaminating breast    cancer cells from hematopoietic progenitor cell preparations using    activation enhanced cell death. Breast Cancer Res Treat. 2002;    72:265.-   8. Hacker D L, Nallet S, Wurm F M. Recombinant Protein Production    Yields from Mammalian Cells: Past, Present, and Future. BioPharm    International. 2008-   9. Yu Y, Niapour M, Zhang Y, Berger S A. Mitochondrial regulation by    c-Myc and hypoxia-inducible factor-1 alpha controls sensitivity to    econazole. Mol Cancer Ther. 2008; 7:483.-   10. Zhang Y, Berger S A. Increased calcium influx and ribosomal    content correlate with resistance to endoplasmic reticulum    stress-induced cell death in mutant leukemia cell lines. J Biol    Chem. 2004; 279:6507.-   11. Prashar Y, Weissman S M. Analysis of differential gene    expression by display of 3′ end restriction fragments of cDNAs. Proc    Natl Acad Sci USA. 1996; 93:659.-   12. Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated    endoplasmic reticulum stress responses in cancer. Cancer Res. 2007;    67:10631.-   13. Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the    inflammatory response. Nature. 2008; 454:455.-   14. Mayer C, Zhao J, Yuan X, Grummt I. mTOR-dependent activation of    the transcription factor TIF-IA links rRNA synthesis to nutrient    availability. Genes Dev. 2004; 18:423.-   15. Xiao L, Grove A. Coordination of Ribosomal Protein and Ribosomal    RNA Gene Expression in Response to TOR Signaling. Curr Genomics.    2009; 10:198.-   16. Mayer C, Bierhoff H, Grummt I. The nucleolus as a stress sensor:    JNK2 inactivates the transcription factor TIF-IA and down-regulates    rRNA synthesis. Genes Dev. 2005; 19:933.-   17. Ota M, Katsuyama Y, Hamano H, Umemura T, Kimura A, Yoshizawa K,    Kiyosawa K, Fukushima H, Bahram S, Inoko H, Kawa S. Two critical    genes (HLA-DRB1 and ABCF1) in the HLA region are associated with the    susceptibility to autoimmune pancreatitis. Immunogenetics. 2007;    59:45.-   18. Wilde D B, Marrack P, Kappler J, Dialynas D P, Fitch F W.    Evidence implicating L3T4 in class II MHC antigen reactivity;    monoclonal antibody GK1.5 (anti-L3T4a) blocks class II MHC    antigen-specific proliferation, release of lymphokines, and binding    by cloned murine helper T lymphocyte lines. J Immunol. 1983;    131:2178.-   19. Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D,    Naldini L. A third-generation lentivirus vector with a conditional    packaging system. J Virol. 1998; 72:8463.-   20. Grynkiewcz G, Poenie M, Tsien R Y. A new generation of Ca2+    indicators with greatly improved fluorescence properties. J Biol    Chem. 1985; 260:3440.-   21. Greco A, Madjar J J. Cell Biology: A Laboratory Handbook,    Vol. 2. J E Celis. 1998-   22. Lievremont J P, Rizzuto R, Hendershot L, Meldolesi J. BiP, a    major chaperone protein of the endoplasmic reticulum lumen, plays a    direct and important role in the storage of the rapidly exchanging    pool of Ca2+. J Biol Chem. 1997; 272:30873.

1.-28. (canceled)
 29. A process for the production of a protein ofinterest comprising: a) increasing production of a protein of interestin a eukaryotic cell according to claim 46; b) culturing the eukaryoticcell under suitable conditions permitting the expression of the proteinof interest and the ABC50 protein or fragment; culturing the cell untilthe protein of interest accumulates and isolating the protein ofinterest.
 30. The process of claim 29 wherein the protein of interest isan antibody or fragment thereof. 31.-33. (canceled)
 34. A vectorcomprising a polynucleotide encoding an ABC50 polynucleotide andoptionally a polynucleotide encoding a protein of interest, wherein thepolynucleotide(s) is/are operably linked to one or more promoters. 35.The vector of claim 34, wherein the vector is a retroviral vector,optionally a lentiviral vector.
 36. A eukaryotic cell a) comprising thevector of claim 34 expressing increased ABC50 protein and/or activitycompared to a control cell not comprising said vector; or b) resistantto Econozole (Ec), wherein the Ec resistant cell has increased ABC50protein levels and/or activity compared to a non-Ec resistant controlcell, wherein the eukaryotic cell is suitable and/or adapted forexpression of a protein of interest.
 37. (canceled)
 38. The cell ofclaim 36, wherein the eukaryotic cell is selected from a yeast, plant,worm, insect, avian, fish, reptile, mammalian, hybridoma, a myeloma cellor a spleen cell.
 39. A system for heterologous protein expressioncomprising the eukaryotic cell of claim 36 b) and an expression vectorcomprising a multicloning site for receiving a heterologouspolynucleotide encoding the heterologous protein to be expressed.40.-42. (canceled)
 43. The method of claim 42, wherein the shRNAcomprises SEQ ID NO: 3 and/or
 4. 44. The method of claim 41 wherein theER stress agent is selected from EC, thapsigargin and tunicamycin.
 45. Acomposition comprising: a polynucleotide comprising SEQ ID NO:3 and/or4, an isolated vector comprising a polynucleotide encoding an ABC50polynucleotide and optionally a polynucleotide encoding a protein ofinterest, wherein the polynucleotide(s) is/are operably linked to one ormore promoters, a eukaryotic cell comprising said vector or a eukaryoticcell that is Ec resistant and has increased protein levels and/oractivity compared to a non-Ec resistant control cell, and/or an isolatedprotein produced according to the method of claim
 46. 46. A method forincreasing the production of a protein of interest, said methodcomprising: I) a) introducing into a eukaryotic cell a polynucleotideencoding an ABC50 protein or active fragment thereof having proteinsynthesis increasing activity and/or eIF2 binding activity andoptionally encoding a selectable marker into a eukaryotic cell, whereinsaid eukaryotic cell expresses a protein of interest or wherein afterthe introduction of the polynucleotide encoding an ABC50 protein oractive fragment the eukaryotic cell is fused to a second eukaryotic cellexpressing a protein of interest, thus producing a eukaryotic cellexpressing ABC50 protein or active fragment thereof and the protein ofinterest, and b) incubating the eukaryotic cell under suitableconditions for a suitable length of time to produce the protein ofinterest in the presence of the ABC50 protein such that the protein ofinterest is produced at higher levels than in the absence of the ABC50protein; II) a), chemically inducing the expression of endogenous ABC50protein in a eukaryotic cell, wherein said eukaryotic cell expresses aprotein of interest or wherein after the chemical induction of thepolynucleotide encoding an ABC50 protein the eukaryotic cell is fused toa second eukaryotic cell expressing a protein of interest, thusproducing a eukaryotic cell expressing ABC50 protein and a protein ofinterest, and b) incubating the eukaryotic cell under suitableconditions for a suitable length of time to produce the protein ofinterest in the presence of the ABC50 protein such that the protein ofinterest is produced at higher levels than in the absence of the ABC50protein; III) a) introducing into a eukaryotic cell a polynucleotideencoding a protein of interest and optionally encoding a selectablemarker, wherein said eukaryotic cell expresses an ABC50 protein oractive fragment thereof having protein synthesis increasing activityand/or eIF2 binding activity, wherein said ABC50 protein or activefragment thereof is recombinantly expressed in the eurkaryotic cellafter the introduction of the polynucleotide encoding an ABC50 proteinor active fragment thereof, thus producing a eukaryotic cell expressingABC50 protein or active fragment thereof and a protein of interest, andb) incubating the eukaryotic cell under suitable conditions for asuitable length of time to produce the protein of interest in thepresence of the ABC50 protein such that the protein of interest isproduced at higher levels than in the absence of the ABC50 protein; IV)a) introducing into a eukaryotic cell a polynucleotide encoding aprotein of interest and optionally encoding a selectable marker, whereinsaid eukaryotic cell expresses an ABC50 protein, wherein said ABC50protein is expressed by chemically inducing the expression of endogenousABC50 protein in the eukaryotic cell, thus producing a eukaryotic cellexpressing ABC50 protein or active fragment thereof and a protein ofinterest, and b) incubating the eukaryotic cell under suitableconditions for a suitable length of time to produce the protein ofinterest in the presence of the ABC50 protein such that the protein ofinterest is produced at higher levels than in the absence of the ABC50protein; V) a) introducing into a eukaryotic cell a polynucleotideencoding an ABC50 protein or active fragment thereof having proteinsynthesis increasing activity and/or eIF2 binding activity andoptionally encoding a selectable marker into a eukaryotic cell, b)introducing into the eukaryotic cell produced in step a) apolynucleotide encoding a protein of interest and optionally encoding aselectable marker, thus producing a eukaryotic cell expressing ABC50protein or active fragment thereof and a protein of interest, and c)incubating the eukaryotic cell under suitable conditions for a suitablelength of time to produce the protein of interest in the presence of theABC50 protein such that the protein of interest is produced at higherlevels than in the absence of the ABC50 protein; or VI) a) chemicallyinducing the expression of endogenous ABC50 protein in a eukaryoticcell, and b) introducing into the eukaryotic cell produced in step a) apolynucleotide encoding a protein of interest and optionally encoding aselectable marker, thus producing a eukaryotic cell expressing ABC50protein or active fragment thereof and a protein of interest, and c)incubating the eukaryotic cell under suitable conditions for a suitablelength of time to produce the protein of interest and the presence ofthe ABC50 protein such that the protein of interest is produced athigher levels than in the absence of the ABC50 protein.
 47. The methodof claim 46, wherein the protein of interest is a heterologous protein.48. The method of claim 46, wherein the method further comprisesisolating the protein of interest, optionally wherein protein ofinterest is secreted and is secreted into a culture medium, the methodfurther comprising isolating the secreted protein from the culturemedium, or wherein the protein of interest is intracellular, the methodfurther comprising lysing the cell and isolating the intracellularprotein of interest, or wherein the protein of interest is membrane orsurface bound, the method further comprising solubilizing the cellmembrane and isolating the membrane protein or surface bound protein ofinterest.
 49. The method of claim 46, wherein the ABC50 proteincomprises SEQ ID NO: 1, 2 or 5; or a protein with at least 85%, 88%,90%, 95%, 99% or 99.5% sequence identity with SEQ ID NO:1, 2 or
 5. 50.The method of claim 46, wherein the increased expression is about 5% toabout 10%, about 11% to about 20%, about 21% to about 30%, about 31% toabout 40%, about 41% to about 50%, 51% to about 60%, 61% to about 70%,71% to about 80%, about 81% to about 90%, about 91% to about 100%, about150% to about 199%, about 200% to about 299%, about 300% to about 499%,or about 500% to about 1000%.
 51. The method of claim 46, wherein theeukaryotic cell is selected from a yeast, plant, worm, insect, avian,fish, reptile and mammalian cell.
 52. The method of claim 46, whereinthe selectable marker is selected from β-galactosidase, chloramphenicolacetyltransferase, firefly luciferase, an antibiotic resistance geneneomycin and hygromycin, dihydrofolate reductase (DHFR) and glutaminesynthetase (GS) and optionally wherein selection comprises amplificationof the integrated DNA by exposure of the selected cells to methotrexate(MTX) or methionine sulphoximine (MSX).
 53. The method of claim 52,wherein the antibiotic resistance gene is selected from neomycin andhygromycin.
 54. The method of claim 46, wherein the ABC50 protein ischemically increased by induction of econazole resistance and selectingfor ABC50 expressing cells.